Drought Resistance Evaluation of Casuarina equisetifolia Half-Sib Families at the Seedling Stage and the Response of Five NAC Genes to Drought Stress

Round 1

Reviewer 1 Report

Introduction.

Ref. 7.8 are very old, it is advisable to replace them with newer ones.

“NAC genes might be associated with the ability of C. equisetifolia to respond to drought stress.” The reason for choosing to study NAC TF genes should be described in detail.

 

Materials and methods.

Please indicate the volume of the pot.

Why not measure the dry weight of plants? This parameter is more objective than height.

 

Discussion.

Subsection 4.2. “This decrement was lower… [32,33].” This statement is incorrect, since the conditions of these experiments were different.

 

References are not formatted according to the rules of Forests.

Subsection 2.1.5. "Seeding" should be corrected to "seedling".

Fig. 3b. “Categery” should be corrected to “category”.

Author Response

Point 1: Ref. 7.8 are very old, it is advisable to replace them with newer ones.

Response 1: We thank you for your comment. We have replaced references 7 and 8 in the original manuscript with a new reference. Please see Ref. 7 in the References in the revised manuscript.

7. Zhang, ; Zhong, C. L.; Chen, Y.; Chen, Z.; Jiang, Q. B.; Wu, C.; Pinyopusarerk, K. Improving drought tolerance of Casuarina equisetifolia seedlings by arbuscular mycorrhizas under glasshouse conditions. New For. 2010, 40(3), 261–271.

Point 2: “NAC genes might be associated with the ability of C. equisetifolia to respond to drought stress.” The reason for choosing to study NAC TF genes should be described in detail.

Response 2: We thank you for your comment. According to your comment, we have supplemented the relevant information. Please see the third paragraph in the Introduction (Page 2 lines 10–15) in the revised manuscript.

“NAC transcription factor (TF)-coding genes represent a large gene family in plants and are key to the drought stress response of plants [18]. The NAC TFs perform specific functions through specific promoter cis-acting elements [19], such as scavenging peroxide in plants [20] and regulating the expression of stress-related genes [21]. In our previous study, we found that NAC TFs may be related to the ability of C. equisetifolia to respond to drought stress (data not yet published).”

Point 3: Indicate the volume of the pot.

Response 3: We thank you for your suggestion. We have specified the volume of the pot in 2.1.2. Sowing and Raising Seedlings (Page 3) in the revised manuscript as follows:

“The dimensions of each pot were as follows: top diameter = 220 mm, bottom diameter = 180 mm, height = 225 mm, tray diameter = 220 mm, and volume = 7 L.”

Point 4: Why not measure the dry weight of plants? This parameter is more objective than height.

Response 4: We thank you for your suggestion. Although we agree that the dry weight of plants can be more objective, but we chose the height to reflect the adaptability of C. equisetifolia to drought stress mainly because we needed to conduct continuous afforestation verification test after the drought stress test to further verify the accuracy of our drought-resistance evaluation results. Therefore, We had to avoid the loss of experimental seedlings due to destructive observation as much as possible.

Point 5: Subsection 4.2. “This decrement was lower… [32,33].” This statement is incorrect, since the conditions of these experiments were different. 

Response 5: We thank you for your constructive comment. We have revised this part in 4.2. Response mechanism of C. equisetifolia to drought stress (Page 13) as follows:

“Our study showed that under long-term drought stress, C. equisetifolia can reduce water consumption by slowing down its growth rate to adapt to the drought environment; in addition, the phylloclades decrease the effective light-receiving area and stomate size, and avoid excessive water loss by wilting.”

Point 6: References are not formatted according to the rules of Forests.

Response 6: We apologize for the oversight. We have formatted the references according to the guidelines of Forests (Page 15).

Point 7: Subsection 2.1.5. "Seeding" should be corrected to "seedling".

Response 7: We apologize for the oversight. We have corrected the mistake in 2.1.5. Observation and statistics (Page 3) in the revised manuscript as follows:

“Seedling preservation rate (P) was then determined.”

Point 8: Fig. 3b. “Categery” should be corrected to “category”.

Response 8: We apologize for the oversight. We have corrected the mistake in Fig. 3b (Page 10).

 

Author Response File: Author Response.docx

Reviewer 2 Report

The topic is original and relevant to the current issues especially in the context of the climate change issues . The paper writing style is clear and easy to read, just need correction and improve the sentences structure in the whole text.  The conclusion presented is well, clear and easy to read and address the main question. 

Some minor English improvement and sentences structure is required in the whole text. 

Author Response

Point 1: The topic is original and relevant to the current issues especially in the context of the climate change issues. The paper writing style is clear and easy to read, just need correction and improve the sentences structure in the whole text.  The conclusion presented is well, clear and easy to read and address the main question. Some minor English improvement and sentences structure is required in the whole text. 

Response 1: We thank you for your positive feedback on our manuscript. We apologize for the language-related errors in our manuscript. Repeated additions and deletions of sentences and sections have led to some errors in English expression and structure to some extent. We have carefully checked the manuscript and fixed the mistakes. Furthermore, the manuscript was checked by native English speakers. These revisions have not affected the content and overall structure of the manuscript. The revised parts are indicated in blue font in the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

The review of manuscript titled "Drought resistance evaluation of Casuarina equisetifolia half-sib families at the seedling stage and the response of five NAC genes to drought stress" by  Huichang Xu, Jinlin Yu, Longhui You *, Shengwu Xiao, Sen Nie, Tuhe Li, Gongfu Ye *, Dichu Lin  

The manuscript by Longhui You, Gongfu Ye  (corresponding authors) and others aimed at investigating the physiological, biochemical and molecular response of  Casuarina equisetifolia to drought stress and expoloration of drought resistance regulation by NAC genes in this species. The authors demonstrated the existence of five drought-tolerant families of C. equisetifolia and determined the variation in physiological and biochemical traits that ensure adequate response and adaptation to drought stress and the relationship of the expression of selected NAC genes to the plants' response to water deficit. In my opinion, the manuscript is interesting and well written; however, it contains some deficiencies which require attention and should be corrected.

 

The Introduction and Materials and Methods sections of the manuscript are written accurately. The first part of the manuscript presents the current state of knowledge in the manuscript topic and the importance of climate change in the expanding problem of water deficit and the emergence and response of plants to drought stress. I believe the problem is the last 10 lines of this Introduction section of the manuscript. This section contains methodological information about the analyses carried out in the paper. However, a description of the aim and hypotheses, which should be at the end of this section, has been omitted.

 

The biological material and methods used in experiments are described precisely. However, subsection 2.2: Data processing and analysis of this section does not contain all the information. How differences between families (drought-resistant families) were analysed. Were only differences between DT and CK tested?

Concerning PCA analysis: Were tests conducted to justify the use of PCA (Bartlett test, KMO test).

 

In the result section of the manuscript, the Figures contain all necessary information.

Subsection 3.1, line 2-3 states that "there was abundant variation which facilitated the selection of drought-resistant families".

However, Table 4 presents overall means for all families, mean standard deviation and mean CV for families. The mean, deviation and CV for individual families are missing. I believe that for clarity of the table and analyses of differences between families, the data for individual families should be completed. It should also be indicated which families differ from which. It would also be useful to mark homogeneous groups within families.

Author Response

Point 1: The Introduction and Materials and Methods sections of the manuscript are written accurately. The first part of the manuscript presents the current state of knowledge in the manuscript topic and the importance of climate change in the expanding problem of water deficit and the emergence and response of plants to drought stress. I believe the problem is the last 10 lines of this Introduction section of the manuscript. This section contains methodological information about the analyses carried out in the paper. However, a description of the aim and hypotheses, which should be at the end of this section, has been omitted.

Response 1: We thank you for your positive comment and valuable suggestion. We have revised the Introduction (Page 2 last 8 lines) as follows:

“In this study, half-sib families of C. equisetifolia were subjected to drought stress, and the changes in growth, morphology, physiological and biochemical indexes, and NAC TF expression in the tested families were analyzed. The response mechanism of C. equisetifolia to drought stress was discussed from a multidimensional scale, and drought-resistant families were preliminarily selected. We focused on the following two aspects: (1) the multidimensional response mechanism of C. equisetifolia to drought stress and (2) the regulatory pathway that might be affected by the five NAC TFs in the drought resistance of C. equisetifolia.”

Point 2: The biological material and methods used in experiments are described precisely. However, subsection 2.3: Data processing and analysis of this section does not contain all the information. How differences between families (drought-resistant families) were analysed. Were only differences between DT and CK tested?

Response 2: We apologize for not clearly explaining how to analyze the differences between families (drought-resistant families). We tested the confidence of the difference between DT and CK using the paired-sample t-test. For the difference between families (drought-resistant families), we calculated the D value of each family using the principal component analysis and membership function method. Using the D value as the variable, the cluster analysis was used to cluster the tested families, and drought resistance was evaluated according to the average D value of each category. We have revised the 2.3. Data processing and analysis (Page 6) as follows:

"Finally, using the D value as the variable, the cluster analysis was used to cluster the tested families, and drought resistance was evaluated according to the average D value of each category."

Point 3: Concerning PCA analysis: Were tests conducted to justify the use of PCA (Bartlett test, KMO test).

Response 3: We thank you for the comment. In our study, H, W, S, MDA, SOD, Pro, GSH, and AsA were tested using the KMO and Bartlett's test. As shown in Table 1, the coefficient of the KMO test was 0.609, and in the Bartlett's test, the P value was <0.001, which indicates that there was a linear correlation in the DC values among the eight indexes and that the principal component analysis can be carried out.

Table 1 KMO and Bartlett's test of DC value of the observation indexes

KMO and Bartlett’s test
Kaiser-Meyer-Olkin Measure of Sampling Adequacy		0.609
Bartlett’s Test of Sphericity	Approx. Chi-Square	118.007
	Df	28
	Sig.	0.000

Point 4: Subsection 3.1, line 2-3 states that "there was abundant variation which facilitated the selection of drought-resistant families". However, Table 4 presents overall means for all families, mean standard deviation and mean CV for families. The mean, deviation and CV for individual families are missing. I believe that for clarity of the table and analyses of differences between families, the data for individual families should be completed. It should also be indicated which families differ from which. It would also be useful to mark homogeneous groups within families.

Response 4: We apologize for the lack of clarity. The results in Table 3 show that the variation coefficients of the eight observation indexes in the DT were between 0.104 and 0.788, which indicates that under drought stress, the variation degree of each observation index of the tested families is high, providing basic conditions for the selection of drought-resistant families. In Table 4, the results of variation coefficient of the DC value of each observation index also confirmed this point (range: 0.104–0.739). Moreover, drought resistance is a functional adaptation to drought stress in plants. It is a complex quantitative trait influenced by several factors. The use of a single index in the analysis limits the results and cannot accurately reflect actual plant drought resistance. Therefore, in this study, eight observation indexes were divided into four comprehensive indexes using the principal component analysis, and the D values of all families were calculated by combining the weight factor and membership function method. Taking the D value as the variable, the families were clustered using cluster analysis, and drought resistance was divided according to the average D value of each category. Therefore, in this study, there was no single multiple comparative analysis of each observation index of the tested families in Tables 3 and 4.

Author Response File: Author Response.docx